Recombinant AAV (rAAV) has proven to be one of the most versatile gene therapy vector systems for a wide range of genetic diseases including cystic fibrosis (CF). However, despite progress in moving this vector system toward clinical trials for CF lung disease, problems associated with both vector design and intracellular barriers to transduction in airway epithelia have hindered success. Over the previous funding period, several important biologic principles of rAAV transduction in polarized human airway epithelia have been established which clarify the present shortcomings of this vector system in current clinical trials. Notable advances in this area of research include the identification that intracellular trafficking to the nucleus is rate limiting in many cell types including polarized airway epithelia. Additionally, the importance of the ubiquitin/proteasome system in intracellular processing of two rAAV serotypes (type 2 and type 5) and the development of pharmacologic approaches to modulate the proteasome and enhance transduction from the apical surface of human airway epithelia (>1000-fold) have been established. However, despite these advances in the identification of efficient proteasome inhibitor cocktails to enhance rAAV transduction from the apical membrane, the underlying mechanisms of their action remain unknown. This proposal rationalizes that a clearer understanding of how the ubiquitin/proteasome system is involved in the intracellular trafficking and processing of rAAV in airway epithelial cells will greatly increase the pace of developing clinically applicable methods for enhancing airway transduction with this vector system. To this end, this grant will seek to clearly define the intracellular pathways of rAAV movement through the cell using both biochemical and imaging techniques. Furthermore, this proposal will attempt to define the predominant endosomal compartment from which rAAV exits into the cytoplasm and the manner in which it subsequently traffics to the nucleus. Finally, this proposal will attempt to elucidate how modulation of the ubiquitin/proteasome system alters intracellular processing of rAAV to promote its movement to the nucleus. Through this research, we hope to define more clearly, how the polarity of airway epithelia limits transduction from the apical surface and the involvement of the proteasome system in this process. A combination of techniques will be used, including co localization of fluorescent-tagged rAAV with GFP-tagged intracellular domains, immuno-affinity isolation of various endosomal compartments using expressed HA-tagged intracellular markers, and expression of dominant negative Rab proteins to inhibit rAAV movement through specific endosomal pathways. Both in vitro and in vivo models of the mouse and human airway will be used to study these pathways. Further elucidation of rAAV transduction biology in the airway will aid in the development of clinically applicable methods for increasing the efficacy of rAAV gene transfer in vivo.